1. Field of the Invention
The present invention relates to an antifungal polypeptide, AlyAFP, obtainable from flowers of plants in the genus Alyssum, and method for controlling pathogenic employing this ant antifungal polypeptide. The antifungal polypeptide can be applied directly to a plant, applied to a plant in the form of microorganisms that produce the polypeptide, or plants themselves can be genetically modified to produce the polypeptide. The present invention also relates to DNA sequences, microorganisms, plants, and compositions useful in these methods.
2. Description of Related Art
A number of plant polypeptides and proteins exhibiting antifungal activity against a variety of plant pathogenic fungi have been isolated (Bowles (1990) Annu. Rev. Biochem. 59:873-907; Brears et al. (1994) Agro-Food-Industry Hi-Tech. 10-13). These antifungal polypeptides and proteins, encompassing several classes including chitinases, cysteine-rich chitin-binding proteins, .beta.-1,3-glucanases, permatins (including zeamatins), thionins, ribosome-inactivating proteins, and non-specific lipid transfer proteins, are believed to play important roles in plant defense against fungal infection.
Recently, another group of plant proteins has been found to function as defensins in combatting infections by plant pathogens (PCT International Publication WO 93/05153). Two small cysteine-rich proteins isolated from radish seed, Rs-AFP1 and Rs-AFP2, were found to inhibit the growth of many pathogenic fungi when the pure protein was added to an in vitro antifungal assay medium. Transgenic tobacco plants containing the gene encoding Rs-AFP2 protein were found to be more resistant to attack by fungi than non-transformed plants.
Proteins similar to radish seed Rs-AFP2 have been isolated from seeds of many other plants (PCT International Publication WO 93/05153; Broekaert et al. (1995) Plant Physiol. 108:1353-1358). All the proteins in this group share similarity in their amino acid sequence, but differ in their antifungal activities against various fungi, especially in the presence of different mono- and divalent salts in the assay medium, which more closely resembles the physiological condition in plant cells: the antifungal activity of some antifungal proteins is dramatically reduced in the presence of 1 mM CaCl.sub.2 and 50 mM KCl (Terras et al. (1992) J. Biol. Chem. 267:15301-15309). The usefulness of an antifungal protein for genetically engineering plant disease resistance can be greatly influenced by the sensitivity of the antifungal activity to salt concentration, since metal ions such K+, Na+, Ca.sup.2+, and Mg.sup.2+ are required for normal physiological functions and are therefore abundantly present in plant cells.
The use of natural protein products to control plant pathogens has been demonstrated, for example, in European Patent Application 0 392 225.